Deadly acceleration in dehydration of Eucalyptus viminalis leaves coincides with high-order vein cavitation

Abstract Xylem cavitation during drought is proposed as a major driver of canopy collapse, but the mechanistic link between hydraulic failure and leaf damage in trees is still uncertain. Here, we used the tree species manna gum (Eucalyptus viminalis) to explore the connection between xylem dysfunction and lethal desiccation in leaves. Cavitation damage to leaf xylem could theoretically trigger lethal desiccation of tissues by severing water supply under scenarios such as runaway xylem cavitation, or the local failure of terminal parts of the leaf vein network. To investigate the role of xylem failure in leaf death, we compared the timing of damage to the photosynthetic machinery (Fv/Fm decline) with changes in plant hydration and xylem cavitation during imposed water stress. The water potential at which Fv/Fm was observed to decline corresponded to the water potential marking a transition from slow to very rapid tissue dehydration. Both events also occurred simultaneously with the initiation of cavitation in leaf high-order veins (HOV, veins from the third order above) and the analytically derived point of leaf runaway hydraulic failure. The close synchrony between xylem dysfunction and the photosynthetic damage strongly points to water supply disruption as the trigger for desiccation of leaves in this hardy evergreen tree. These results indicate that runaway cavitation, possibly triggered by HOV network failure, is the tipping agent determining the vulnerability of E. viminalis leaves to damage during drought and suggest that HOV cavitation and runaway hydraulic failure may play a general role in determining canopy damage in plants.


Supplemental Figure S4
Example of plot of stomatal conductance (gs, mmol m -2 s -1 ) through time (s) for one leaves of E. viminalis. The red box shows the steady state phase (when stomata are closed) that was used to calculate gmin (mmol m -2 s -1 ).

Supplemental Figure S5
Changes to leaf fluorescence (Fv/Fm) (A, n=6) and width (B, n=13) during time (Min). Solid black lines are regressions with 95% confidence intervals (light grey shading). Red solid lines represent the mean time at the slope breakpoint in Fv/Fm and leaf width respectively, and dashed lines represented the std associated. Figure S6 Leaf water potential inducing a 50% loss of water transport capacity (P50, MPa) in the midrib, major and hov (high order veins) of E. viminalis leaves (n=13).

Supplemental
Different letters indicate significant differences between orders (p-value <0.05). The horizontal line in boxes is the median value, and vertical lines are the 25th and 75th percentiles. Figure S7 The relationship between the breakpoint in leaf width and 20% HOV cavitation, suggesting a tight connection in time and water potential. (A) The relationship between the time at which we observed the breakpoint on leaf width to occur (Time BP Leaf width, Min) and the time at 20% high order vein cavitation (Time 20% HOV cavitation, Min) for each individual of E. viminalis (n=13) is described by a strong linear correlation (R 2 = 0.98, pvalue <0.05). The distance of the residuals to the 1:1 relationship is -17.92 ± 88.96 Min. (B) The relationship between the at which we observed the breakpoint on leaf width to occur (BP Leaf Width, MPa) and the Ψ stem at 20% high order vein cavitation (P20 HOV, MPa) for each individual of E. viminalis (n=13) is described by a strong linear correlation (R 2 = 0.93, p-value <0.05). The distance of the residuals to the 1:1 relationship is 0.01 ± 0.13 MPa. Dotted black lines indicate 1:1 relationship.

Supplemental Figure S8
Relationship between the time at which breakpoint in leaf width occur (BP Leaf Width, Min) and the time at which 20% of midrib cavitates (A) and the 50% of midrib cavitates (B), with the distance of the points to the 1:1 relationship been respectively -459.58 ± 261.57  Min. Relationship between the Ψ stem at which breakpoint in leaf width occur (BP Leaf Width, Min) and the Ψ stem at which 20% of midrib cavitates (A) and the 50% of midrib cavitates (B). The distance of the residuals to the 1:1 relationship si respectively 0.79 ± 0.50 MPa and 0.54 ± 0.53 MPa. R 2 values are reported and dotted black lines indicate 1:1 relationship.

Supplemental Method S1
Point of runaway vulnerability with full sigmoidal curve Let our vulnerability curve be described using the sigmoidal function where K is the hydraulic conductance (in mmol m -2 s -1 MPa -1 ), K max is the maximum hydraulic conductance (also in mmol m -2 s -1 MPa -1 ), φ is the negative of water potential (so that water potential is represented by a positive quantity, in MPa), P 50 is the φ where K is half maximum (also in MPa) and α is a width parameter (again, in MPa).
The liquid flux of water (J, in mol m -2 s -1 ) is then given by where φ s is the negative of source water potential (in MPa).
Liquid and vapour fluxes of water are equal at steady state. If we consider the case of a constant cuticular transpiration (E c , in mmol m -2 s -1 ), then the steady state occurs when (3) We are interested in finding the point of runaway vulnerability for the case of a constant cuticular transpiration (E c , in mmol m -2 s -1 ). The point of runaway vulnerability happens when J is a maximum (J max ) and is equal to E c . This is easiest to see graphically by plotting J and E c and observing where they intersect (the steady states). If J max > E c then two steady states exist (one stable, one unstable), but the two solutions converge as either E c or φ s increases. At the point of runaway vulnerability, only one solution exists and J max = E c . If E c or φ s increases further, so that J max < E c , then no steady state exists and runaway vulnerability occurs. For one of E c or φ s given, there is a unique value for the other, and hence a value for φ, where runaway vulnerability occurs. Now, J max occurs when = 0.

This gives
This equality holds at the point of runaway cavitation. We can eliminate one variable and substitute it back into equation 3 and solve for the other variable. As we want an equation for what φ runaway vulnerability occurs at for a given E c , we will eliminate φ s : This gives the negative of water potential where runaway vulnerability will first occur.
We might also want to know what percentage loss of K this corresponds to. If we let the percentage loss of K be x, then This gives the percentage loss of hydraulic conductance where runaway cavitation should occur.